Selective acylation of secondary hydroxyl groups

ABSTRACT

The invention provides methods and compositions for selectively acylating a specific hydroxyl group in a molecule of interest containing at least two unprotected secondary hydroxyl groups. Although the methods and compositions of the invention have general applicability, they are particularly useful in the selective acylation of the taxane molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

The application claims priority from U.S. Provisional application Ser.No. 60/370,252, filed Apr. 5, 2002. This application is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to methods and compositions forselectively acylating a secondary hydroxyl group of a compoundcomprising a plurality of unprotected secondary hydroxyl groups. Morespecifically, the invention relates to methods and compositions forselectively acylating the C-2′ hydroxyl group of a taxane moleculecontaining an unprotected C-7 hydroxyl group.

BACKGROUND OF THE INVENTION

Acylation is a process frequently used in the chemical arts to introduceone or more carbonyl-containing substituents into a compound ofinterest, often to protect hydroxyl or amine groups. Over the years,investigators have identified a variety of reagents and reactionconditions for acylating particular functionalities. Of particularinterest is the ability to selectively acylate a desired functionalitywithout the need to protect and deprotect other reactive sites therebycreating a more efficient synthetic scheme.

For example, Ishihara et al. describe a method for selectively acylatinga primary alcohol in presence of secondary alcohols using an acylchloride (Ishihara et al. (1993) J. Org. Chem. 58: 3791-3793). In anexemplary reaction scheme, a 1:1 mixture of 1-octanol and 2-octanol inmethylene chloride was reacted with acetyl chloride in the presence ofvarious bases, for ex ample, 2,4,6-collidine, N,N-diisopropylethylamine,or 1,2,2;6,6-pentamethylpiperidine. Under the conditions described, theprimary alcohol in 1-octanol was acylated preferentially over thesecondary alcohol in 2-octanol.

Garegg et al. similarly describe the regioselective acetylation of theprimary alcohols of a tetrasaccharide in the presence of its secondaryalcohols (Garegg (1997) J. Carbohydr. Chem. 16(7): 973-981).Specifically, Garegg et al. utilize an excess of acetyl chloride andcollidine in methylene chloride at low temperature (−70° C.) to performthe selective acetylation.

In another example, Szeja identified reaction conditions to selectivelyacylate a secondary hydroxyl group at either the 2-position or the3-position in a sugar molecule where both hydroxyl groups arc initiallyunprotected (Szeja (1979) Synthesis 821-822). In the paper, Szejareports that methyl 4,6-O-benzlidene-a-D-galactopyranoside was partiallyesterified in 46% yield at its more reactive 3-hydroxyl group usingbenzoyl chloride and pyridine (see, e.g., Haines (1976) Adv. Carbohydr.Chem. Biochem. 33: 11). However, Szeja discloses the regioselectivebenzoylation of methyl 4,6-O-benzlidene-a-D-galactopyranoside at eitherits 2-hydroxyl group or its 3-hydroxyl group via phase transfercatalysis.

More specifically, when benzoyl chloride was added to methyl4,6-O-benzlidene-a-D-galactopyranoside in benzene andhexamethylphosphoric triamide with tetrabutylammonium chloride and a 40%aqueous sodium hydroxide solution forming the aqueous phase, thereaction provided the 3-O-benzoyl product in a 62% yield. In contrast,when benzoyl chloride was added to methyl4,6-O-benzlidene-a-D-galactopyranoside in the same phase transfercatalysis system without the hexamethylphosphoric triamide present, thereaction provided the 2-O-benzoyl product in a 69% yield.

Regarding the acylation of taxanes, U.S. Pat. Nos. 5,319,112 and5,470,866 describe reaction conditions for acylating the hydroxyl groupsat the C-2′ positions of the taxane molecules paclitaxel anddihydrocephalomannine. In the reactions, a mixture of paclitaxel anddihydrocephalomannine dissolved in acetonitrile was combined withbenzoic acid, dicyclohexylcarbodiimide and4-(N,N-dimethylamino)pyridine. The reaction resulted in benzoylation ofthe C-2′ position of paclitaxel and dihydrocephalomannine. Taxanemolecules acylated at the C-2′ position reportedly are useful startingmaterials for producing oxalate and oxamido derivatives of taxanes (seU.S. Pat. No. 5,470,866).

Paclitaxel, also known as taxol A (TAXOL® being a registered trademarkof the Bristol-Myers Squibb Company), is a member of the taxane family,and is a naturally occurring diterpenoid. Paclitaxel has been shown tohave great value as an anti-cancer drug. Paclitaxel can be isolated fromcertain yew trees, for example, Taxus brevifolia, and certain species ofTaxus media (e.g., species known as “Hill,” “Hicksii” and “dark greenspreader”) (see U.S. Pat. No. 5,744,333), extracted from cell cultures,or synthesized completely or partially in vitro. Notwithstanding thesemethods, the global supply of paclitaxel has been quite limited, andthere is an ongoing need for other methods for producing paclitaxel costeffectively on a larger scale.

Because of the promising clinical activity of certain taxanes (e.g.,paclitaxel) against various types of cancer, there is an ongoing needfor different methods for preparing paclitaxel and other taxanemolecules, including paclitaxel derivatives and analogues. There also isa need for paclitaxel derivatives having a range of in vivo and in vitroactivities, as well as paclitaxel derivatives having similar biologicalactivities to paclitaxel. It is believed that the preparation ofpaclitaxel analogues may result in the synthesis of compounds withcomparable or greater potency, superior bioavailability, or fewer sideeffects than paclitaxel. In support of this approach, a paclitaxelanalogue known as docetaxel (TAXOTERE®) has been identified.

Docetaxel, which differs from paclitaxel only in the nature of theN-acyl substituent and the absence of a 10-acetyl group, is reported tobe twice as active as paclitaxel in certain assays (see U.S. Pat. No.5,319,112). It is contemplated that other paclitaxel derivatives not yetidentified may have other beneficial and pharmacologically desirableproperties. Furthermore, there is also need for paclitaxel analoguesthat can be used as taxane standards. For example, it is desirable tomake easily synthesizable analogues that can be used for characterizingstructure-activity relationships of taxane molecules, as chromatographystandards, or starting or intermediate molecules in the synthesis ofvarious other taxane molecules.

SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositions forselectively acylating a secondary hydroxyl group of interest in acompound that contains a plurality of unprotected secondary hydroxylgroups using an acid halide and a hindered base. Although the inventiondescribed herein has general applicability in the chemical arts, it hasparticular applicability in the synthesis of taxane molecules.

In one aspect, the invention provides a method for selectively acylatinga compound comprising at least two secondary hydroxyl groups that areunprotected. The method generally comprises the steps of providing asolution of the compound of interest in a suitable solvent andcontacting the solution with a base (e.g., hindered base) and anacylating agent (e.g., acid halide) thereby to selectively acylate onesecondary hydroxyl group relative to at least one other secondaryhydroxyl group. Alternatively, the acylating agent may be a carboxylicacid anhydride or a carboxylic acid in conjunction with a coupling agentsuch as dicyclohexylcarbodiimide orN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide. Other hydroxyl acylatingagents known in the art may also be used in the present invention. Alist of these alternatives is described in J. W. Barton, “ProtectingGroups in Organic Chemistry,” J. G. W. McOmie, ed., Plenum Press, NewYork, N.Y., 1973, and in T. W. Greene and P. G. M. Wuts, “ProtectiveGroups in Organic Synthesis,” John Wiley & Sons, New York, N.Y., 1999.In the present invention, the compound to be acylated may be anymolecule that comprises at least two secondary hydroxyl groups. In apreferred embodiment, such compound is a taxane molecule.

In a preferred embodiment, the acid halide is an acid chloride and thehindered base is a trialkylamine or a pyridine derivative. Preferredacid chlorides include benxoyl chloride, tigloyl chloride, hexanoylchloride, butyryl chloride, 2-methylbutyryl chloride, phenylacetylchloride, furoyl chloride, and tert-butyl chloroformate. Preferredtrialkylamines include N-ethyldicyclohexylamine,NN-diisopropylethylamine, and tripropylamine. Preferred pyridinederivatives include 2,6-lutidine, 2,4,6-collidine,2,3,5,6-tetramethylpyridine, 2,6-di-tert-butylpyridine, and2,6-di-tert-butyl-4-(dimethylamino) pyridine or 2,6-dimethyl-4(dimethylamino)pyridine.

In preferred embodiments of the above-described methodology, theinvention provides a method of selectively acylating a hydroxyl grouplocated at the C-2′ position of a taxane molecule while the hydroxylgroup located at the C-7 position of the taxane molecule is unprotected.The method comprises the steps of providing a solution comprising ataxane molecule in an organic solvent and contacting the solution with ahindered base and an acid halide thereby to acylate the hydroxyl grouplocated at the C-2′ position of the taxane molecule. The acid halidesand hindered bases described above are applicable to this preferredembodiment.

The selective acylation process preferably occurs within six hours, ormore preferably within four hours, after addition of the acid halide andthe hindered base to a compound of interest. Additionally, the selectiveacylation reaction preferably occurs at a temperature of about 40° C. orless, or more preferably at about ambient temperature, e.g., from about20° C. to about 25° C.

The organic solvent used in the selective acylation reaction of a taxanemolecule typically is an aprotic solvent that solubilizes a taxanemolecule at a concentration of at least about 10% by weight, andpreferably at least about 15% by weight

Tetrahydrofuran is the Preferred Organic Solvent

The foregoing, and other features and advantages of the invention, aswell as the invention itself, will be more fully understood from thedescription, figures, and claims which follow.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that a secondary hydroxyl group can beselectively acylated in the presence of other unprotected secondaryhydroxyl groups using an acid halide and a hindered base. The methods ofthe invention may permit selective acylation to occur in a relativelyshort time and/or under relatively mild reaction conditions. Byexploiting methods of the invention, it is possible to eliminateprotection/deprotection steps in the overall synthetic sequence of atarget compound, e.g., a biologically active natural product. Inparticular, the method of the invention has utility in the synthesisand/or transformation of taxane molecules.

As used herein, an “acid halide” is a compound which contains a carboxyhalide functionality, i.e., —C(O)X, where X is a halogen such asfluorine, chlorine, bromine, or iodine. An acid halide can include analkoxy group, an alkyl group, an aryl group, an arylalkyl group, anester group, an ether group, a heterocyclic group, a vinyl group, andcombinations thereof. An acid halide also may be substituted withsubstituents such as alkanoyloxy groups, alkenyl groups, alkylsilylgroups, alkylsulfonyl groups, alkylsulfoxy groups, alkylthio groups,alkynyl groups, amino groups such as mono- and di-alkylamino groups andmono- and di-arylamino groups, amide groups, carboxy groups,carboxyalkoxy groups, carboxyamide groups, carboxylate groups, haloalkylgroups, halogens, hydroxyl groups, nitrile groups, nitro groups,phosphate groups, siloxy groups, sulfate groups, sulfonamide groups,sulfonyloxy groups, and combinations of these. Preferred acid halidesare acid chlorides such as benzoyl chloride, tigloyl chloride, hexanoylchloride (caproyl chloride), butyryl chloride, 2-methylbutyryl chloride,phenylacetyl chloride, furoyl chloride, and tert-butyl chloroformate.

As used herein, an “acyl group” means a linear, branched, or cyclicsubstituent having a carbonyl group which is attached to either anoxygen atom, e.g., of a hydroxyl group, or a nitrogen atom, e.g., of anamino group. An acyl group can include an alkoxy group, an alkyl group,an aryl group, an arylalkyl group, an ester group, an ether group aheterocyclic group, a vinyl group, and combinations thereof. An acylgroup also may be substituted with substituents such as alkanoyloxygroups, alkenyl groups, alkylsilyl groups, alkysulfonyl groups,alkylsulfoxy groups, alkylthio groups, alkynyl groups, amino groups suchas mono- and di-alkylamino groups and mono- and di-arylamino groups,amide groups, carboxy groups, carboxyalkoxy groups, carboxyamide groups,carboxylate groups, haloalkyl groups, halogens, hydroxyl groups, nitrilegroups, nitro groups, phosphate groups, siloxy groups, sulfate groups,sulfonamide groups, sulfonyloxy groups, and combination of these. Itshould be understood that an acyl group also can be an amino protectinggroup or a hydroxyl protecting group. As a hydroxyl protecting group, anacyl group may form an ester or carbonate. As an amino protecting group,an acyl group may form an amide or a carbamate. Examples of acyl groupsinclude, but are not limited to, alkoyl groups, aroyl groups, arylalkoylgroups, vinoyl groups. Preferred acyl groups are benzoyl, ethanoyl,tigloyl, or 2-methyl-2-butenoyl, 2-methyl-1-propenoyl, hexanoyl, butyrl,2-methybutyryl, phenylacetyl, propanoyl, furoyl, andtert-butyloxycarbonyl.

As used herein, an “acylating agent” includes any agent that whenreacted with a hydroxyl group or amine group yields an ester carbonateamide or carbanate. Acylating agents may include, but are not limitedto, a carboxylic acid anhydride or a carboxylic acid in conjunction witha coupling agent such as dicyclohexylcarbodiimide orN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide. Other hydroxyl acylatingagents known in the art may also be used in the present invention. Alist of these alternatives is described in J. W. Barton, “ProtectingGroups in Organic Chemistry,” J. G. W. McOmie, ed., Plenum Press, NewYork, N.Y., 1973, and in T. W. Greene and P. G. M. Wuts, “ProtectiveGroups in Organic Synthesis,” John Wiley & Sons, New York, N.Y., 1999.

As used herein, an “alkoxy group” means a linear, branched, or cyclicsaturated hydrocarbon attached to an oxygen atom. Preferably, an alkoxygroup has between one and six carbon atoms. An alkoxy group also refersto substituted alkoxy groups, which may include substituents such asalkanoyloxy groups, alkenyl groups, alkyl groups, alkylsilyl groups,alkylsulfonyl groups, alkylsulfoxy groups, alkylthio groups, alkynylgroups, amino groups such as mono- and di-alkylamino groups and mono-and di-arylamino groups, amide groups, aryl groups, arylalkyl groups,carboxy groups, carboxyalkoxy groups, carboxyamide groups, carboxylategroups, haloalkyl groups, halogens, hydroxyl groups, nitrile groups,nitro groups, phosphate groups, siloxy groups, sulfate groups,sulfonamide groups, sulfonyloxy groups, and combinations of these.Preferred examples of alkoxy groups include, among others, methoxy,ethoxy, propoxy, cyclopropoxy, isopropoxy, n-butoxy, isobutoxy,sec-butoxy, tert-butoxy, cyclobutoxy, pentoxy, isopentoxy, neo-pentoxy,cyclopentoxy, hexoxy, and cyclohexoxy.

As used herein, an “alkyl group” means a linear, branched, or cyclicsaturated hydrocarbon. Preferably, an alkyl group has between one andsix carbon atoms. An alkoxy group also refers to substituted alkylgroups, which may include substituents such as alkanoyloxy groups,alkenyl groups, alkoxy groups, alkylsilyl groups, alkylsulfonyl groups,alkylsulfoxy groups, alkylthio groups, alkynyl groups, amino groups suchas mono- and di-alkylamino groups and mono- and di-arylamino groups,amide groups, aryl groups, arylalkyl groups, carboxy groups,carboxyalkoxy groups, carboxyamide groups, carboxylate groups, haloalkylgroups, halogens, hydroxyl groups, nitrile groups, nitro groups,phosphate groups, siloxy groups, sulfate groups, sulfonamide groups,sulfonyloxy groups, and combinations of these. Preferred substituentsare alkoxy groups, amino groups such as dialkylamino groups, diarylaminogroups, carboxylic acid-containing groups, haloalkyl groups, halogens,hydroxyl groups, nitrile groups, nitro groups, and sulfonic acid groups.Examples of preferred alkyl groups include, but are not limited to,methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl, neo-pentyl,1-ethylpropyl, cyclopentyl, hexyl, and cyclohexyl.

As used herein, an “amino protecting group” means a substituent of anamino group that is employed to block or protect the aminofunctionality, often while reacting other functional groups on themolecule. Example of amino protecting groups are well known in the artand are described in J. W. Barton, “Protecting Groups in OrganicChemistry,” J. G. W. McOrnie, ed., Plenum Press, New York, N.Y., 1973,and in T. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” John Wiley & Sons, New York, N.Y., 1999. It should beunderstood that an amino protecting group may remain on an end product.

As used herein, an “aryl group” means a phenyl group or naphthyl group,which is optionally substituted. Examples of substituents on aryl groupsinclude, but are not limited to, alkanoyloxy groups, alkenyl groups,alkoxy groups, alkylsilyl groups, alkylsulfonyl groups, alkylsulfoxygroups, alkylthio groups, alkynyl groups, amino groups such as mono- anddi-alkylamino groups and mono- and di-arylamino groups, amide groups,aryl groups, arylalkyl groups, carboxy groups, carboxyalkoxy groups,carboxyamide groups, carboxylate groups, haloalkyl groups, halogens,hydroxyl groups, nitrile groups, nitro groups, phosphate groups, siloxygroups, sulfate groups, sulfonamide groups, sulfonyloxy groups, andcombinations of these. Preferred substituents are alkoxy groups, alkylgroups, amino groups such as dialkylamino groups, diarylamino groups,carboxylic acid-containing groups, haloalkyl groups, halogens, hydroxylgroups, nitrile groups, nitro groups, and sulfonic acid groups.

As used herein, an “arylalkyl group” means an aryl group attached to analkyl group. An example of an arylalkyl group is a benzyl group.

As used herein, a “basic baccatin m structure” means a compound havingthe formula:

where each of R₁, R₂, R₄, R₇, and R₁₀ independently is hydrogen, analkyl group, an acyl group, an aryl group, an arylalkyl group, a vinylgroup, an ether group, an ester group, a glycoside group, an oxo-group,or a hydroxyl protecting group. Included within the definition of abasic baccatin III structure is baccatin III, which has the formula:

and 10-deacetylbaccatin III, which has the formula:

where Ac is an acetyl or acetate group (CH₃C(O)—), and Bz is a benzoylgroup (PhC(O)— or C₆H₅C(O)—)

As used herein, an “ester group” means a linear, branched, or cyclicsubstituent having an ester functionality, i.e., —C(O)O—. Examples ofester groups include acyl groups such as ethanoyl and propanoyl, whichare bound to a hydroxyl group.

As used herein, an “ether group” means a linear, branched, or cyclicsubstituent having an ether functionality, i.e., —COC—. An examples ofan ether group includes, but are not limited to, HOCH₂CH₂OC(CH₂OH)H—.

As used herein, a “glycoside group” or a “glycosyl group” means any of anumber of sugar derivatives that contain a non-sugar group bonded to anoxygen or nitrogen atom and that on hydrolysis yield a sugar such asglucose. An example of a preferred gylcosyl group is xylosyl.

As used herein, a “halogen” means fluorine, chlorine, bromine, and/oriodide.

As used herein, a “heterocyclic group” is a saturated, unsaturated, oraromatic cyclic compound that contains at least one atom other thancarbon, e.g., oxygen, nitrogen, or sulfur, in a ring. Examples ofheterocyclic groups include furyls such as 2-furan, morpholino,piperidino, piperazino, N-methylpiperazino, pyrrollyl, pyridyl, andthiophene.

As used herein, the term “hindered base” includes any base that has athree-dimensional structure or contains one or more substituents whichprevents the base or a reactive intermediate thereof from reacting withat least one of at least two unprotected secondary hydroxyl groupspresent in a molecule. In one alternative embodiment, the hindered baseis a trialkylamine where at least one of the alkyl groups is larger thanethyl, or a pyridine derivative that is substituted at least at its2-position, e.g. 2-ethylpyridine. Preferably, the hindered base is atrialkylamine having two of its alkyl groups larger than ethyl, or apyridine derivative that is substituted at least at its 2- and6-positions include, but are not limited to, alkyl groups, alkoxygroups, and halogens. Examples of hindered bases include, but notlimited to, 2,3,5-collidine, 2,4,6-collidine, 2,6-di-tert-butylpyridine,2-6-di-tert-butl-4-(dimethylamino)pyridine, 2,6di-methyl-4-(dimethylamino)pyridine, 2,3-lutidine, 2,4-lutidine,2,5-lutidine, 2,6-lutidine, 2,3,5,6-tetramethylpyridine,N-tert-butylmorpholine, N,N-diisopropylethylamine,N,N-diisopropylisobutylamine, N-ethyldicyclohexylamine, triethylamine,triisoproplyamine, tripropylamine, imidazole,1,5-diazabicylo[4.3.0]non-5-ene (DBN), 1,4-diazabicylo[2.2.2.]octane(Dabco™), 1,8-diazabicylo[5.4.0]undec-7-ene (DBI).

As used herein, a “hydroxyl protecting group” means a substituent of ahydroxyl group that is employed to block or protect the hydroxylfunctionality, often while reacting other functional groups on themolecule. Examples of hydroxyl protecting groups are well known in theart and are described in J. W. Barton, “Protecting Groups in OrganicChemistry,” J. G. W. McOmie, ed., Plenum Press, New York, N.Y., 1973,and in T. W. Green and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” John Wiley & Sons, New York, N.Y., 1999. It should beunderstood that a hydroxyl protecting group may remain on an endproduct. Examples of preferred hydroxyl protecting groups include, amongothers, acyl groups such as acetate (Ac) and benzoyl (Bz),trimethylsilyl (TMS), triethylsilyl (TES), trichloroethoxycarbonyl,2,2,2-trichloroethyloxycarbonyl (Cl₃CH₂OC(O)—).

As used herein, an “oxo-group” means a substituent derived from theoxidation of a glycoside group such as a xyloside as described in U.S.Pat. No. 5,356,928.

As used herein, each of “selective acylation,” “selectively acylating,”and “selectively acylated” means that about 85% or greater of the endproducts is acylated at the desired secondary hydroxyl groups(s) withoutacylation of at least one other secondary hydroxyl group, regardless ofthe overall yield of the reaction. Preferably, about 90% or greater ofthe end products is acylated at the desired secondary hydroxyl groupand/or about 5% or less of a bis- or higher order acylated product isformed and/or about 1% or less of the starting animal, i.e., thecompound to be selectively acylated, remains unreacted. More preferably,about 95% or greater of the end products is acylated at the desiredsecondary hydroxyl group and/or about 2% or less of a bis- or higherorder acylated product is formed and/or about 0.1% or less of thestarting material remains unreacted.

As used herein, “taxane molecule” means a molecule that contains a basicbaccatin III structure with a (2R,3S)-C₆H₅CH(Rx)CH(OH)C(O)— groupforming an ester with the hydroxyl group located at the C-13 position ofthe basic baccatin III structure (where the reference numerals used todesignate atom positions on a taxane molecule are in accordance withstandard taxane nomenclature). The group represented by Rx can be anamino group, a salt of an amino group (e.g., an ammonium salt), an aminogroup which is protected with an amino protecting group, or asubstituent which may be converted into an amino group. Various isomers,homologues, and analogues of the basic baccatin III structure, and ofthe (2R,3S)-C₆H₅CH(Rx)CH(OH)C(O)— group also are included within thedefinition of a taxane molecule. For example, a 10-deacetylbaccatin IIIstructure is contemplated within the scope of a taxane molecule.Included within the definition of a taxane molecule are taxol A(paclitaxel), taxol B (cephalomannine), taxol C, taxol D, taxol E, taxolF, taxol G, docetaxel (TAXOTERE®), and nonataxel (see, e.g., Table 1).

As used herein, a “vinyl group” means a linear or branched substituenthaving a carbon-carbon double bond. Examples of vinyl groups include,but are not limited to, 1-methyl-1-propenyl (CH₃CH═C(CH₃)—) and2-methyl-1-propenyl ((CH₃)₂C═CH—). TABLE 1 Examples of R_(N) Groups InKnown Taxane Molecules Taxane R_(N)— Molecular Structure taxol A(paclitaxel)

taxol B (cephalomannine)

taxol C

taxol D

taxol E

taxol F

taxol G

Docetaxel (Taxotere ®)

Nonataxel

In the present invention, the taxane compound acylated in accordancewith the present invention may be derived from any Taxus species,including but not limited to, Taxus brevifolia, T. baccata (Europeanyew), T. cuspidata, T. globosa (Mexican yew), T. floridana (Floridayew), T. canadenisis (Canadian yew), T. wallichiana (Himalayan yew), T.yunnanensis, T. chinensis, and also a number of ornamental hybrids, suchas T.x media cultivars, e.g.: T.x media ‘Densiformis’, T.x ‘Hicksii’,T.x ‘Brownii’, T.x ‘Dark Green Spreader’, T.x ‘Runyan’, T.x ‘Wardii’,T.x ‘Tautonii’, T. cuspidata ‘Capitata’, etc. Among Taxus species, Taxusx media cultivars are particularly preferred. For example, preferredcultivars include, but are not limited to, T.x ‘Hicksii’ or T.x ‘DarkGreen Spreader’. Further, the starting taxane compound may be derivedfrom grown plant cells, or culture supernatants obtained by using invitro culture technology. The starting taxane compound may also beobtained from semi-synthesis or total synthesis procedures.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present invention also consistessentially of, or consist of, the recited components, and that theprocesses of the present invention also consist essentially of, orconsist of, the recited processing steps. Further, it should beunderstood that the order of steps or order for performing certainactions are immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

It should be understood that in addition to acid halide, a hindered baseand one solvent, a selective acylation reaction of the invention mayemploy additional reagents including additional hindered or unhinderedbases, additional solvents and/or solvent systems, and/or otherchemicals and reagents not affecting the selective acylation reaction.

In one aspect, the invention relates to a method of selectivelyacylating a compound that contains at least two unprotected secondaryhydroxyl groups using an acid halide and a hindered base. The methodgenerally includes the steps of providing a solution of the compound ina solvent and contacting the solution with a hindered base and an acidhalide whereby at least one secondary hydroxyl group is selectivelyacylated in preference to another secondary hydroxyl group.

Various acid halides may be used in the practice of the invention, e.g.acid fluorides, acid chlorides, acid bromides, and acid iodides.Depending on the particular end product and/or any reaction conditionconstraints, e.g., a temperature sensitive molecule, choice of asuitable acid halide is well within the skill of the art. Preferred acidhalides include, among others, benzoyl halide, tigloyl halide, hexanoylhalide (caproyl halide), butyryl halide, 2-methylbutyryl halide,phenylacetyl halide, furoyl halide, and tert-butyl haloformate. However,acid chlorides are preferred.

Various hindered bases may be used in the practice of the invention,although trialkylamines and pyridine derivatives are preferred. As withthe choice of an acid halide, a skilled artisan, with the knowledge ofthe invention, readily would be able to identify a particular hinderedbase suitable for the selective acylation of a specific molecule. Inparticular, the choice of hindered base depends on the spacial locationand steric environment of the secondary hydroxyl groups of the startingmaterial. Hindered bases include, but are not limited to,2,3,5-collidine, 2,4,6-collidine, 2,6-di-tert-butylpyridine,2-6-di-tert-butl-4-(dimethylamino)pyridine,2,6-dimethyl-4-(dimethylamino)pyridine, 2,3-lutidine, 2,4-lutidine,2,5-lutidine, 2,6-lutidine, 2,3,5,6-tetramethylpyridine,N-tert-butylmorpholine, N,N-diisopropylethylamine,N,N-diisopropylisoutylamine, N-ethyldicyclohexylamine, triethylamine,triisoproplyamine, tripropylamine, imidazole,1,5-diazabicylo[4.3.0]non-5-ene (DBN), 1,4-diazabicylo[2.2.2.] octane(Dabco™), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Preferred hinderedbases are 2,4,6-collidine, 2.6-lutidine, N,N-diisopropylethylamine,N-ethlydicyclohexylamine, tripropylamine.

The number of molar equivalents of acid halide and hindered base used ina selective acylation reaction based on the moles of starting materialcan vary depending on the choice of acid halide and hindered base aswell as other desired reaction parameters and conditions. In theory, itmay be necessary to use only one equivalent of an acid halide to avoidbis- or higher order acylation products. However, excess acid halide ispreferred to ensure complete reaction of the starting material with thegreatest percent yield of end product and/or to increase the rate ofreaction.

When using an excess of acid halide and/or hindered base, selectivity ofthe acylation should not be sacrificed or it should be within tolerablelimits. Preferably greater than 2 equivalents, or greater than about 3equivalents, of each of an acid halide and hindered base are used. Morepreferably, about 4 equivalents or more of each acid halide and hinderedbase are used. In certain embodiments, the number of equivalents of theacid halide is not equivalent to the number of equivalents of thehindered base. In a preferred embodiment, the number of equivalents ofthe acid halide is substantially similar to the number of equivalents ofhindered base.

Regarding complete reaction of the starting material, for commercialapplications, preferably less about 3%, or less than about 1%, of thestarting material remains after the selective acylation reaction, andmore preferably, less than about 0.1% remains. Further, with respect togeneration of unwanted side products, preferably less than about 3%, orless than about 2%, of the bis- or higher order acylation product isformed, and more preferably less than about 1% is formed.

Without wishing to be bound to any particular theory, when the hinderedbase is a pyridine derivative, the reaction is believed to proceedthrough a positively charged acyl pyridinium reactive intermediate.Accordingly, if an excess of acid halide is used, an excess of thehindered base also should be used so that an excess of the acylpyridinium intermediate is formed thereby facilitating the completereaction of the starting material. If other hindered bases also proceedthrough reactive intermediates with an acid halide, then an excess ofthe hindered base should be used. However, because the reaction mayproceed through an acyl/hindered base intermediate, it may beunnecessary to have greater than one equivalent of the hindered basepresent as only one equivalent of acid halide should be used up in thereaction with the starting material so that only one equivalent of thehindered base is necessary to scavenge the free halide. Regardless, thedetermination of an appropriate number of equivalents of acid halide andhindered base to use in a particular selective acylation reaction isconsidered to be within the level of skill in the art.

Other reaction parameters and conditions may depend on a variety offactors, e.g., the particular starting material, the spatial locationand steric environment of the secondary hydroxyl groups to beselectively acylated, the spatial location and steric environment ofother secondary hydroxyl groups, and the choice of hindered base andacid halide.

In one alternative embodiment, a suitable solvent may be aprotic,substantially anhydrous, and able to dissolve or solubilize the startingmaterial(s) and acylating reagents to a sufficient degree so that ahigher throughput of end product may be realized and excess amounts ofsolvents can be avoided. However, in all applications, each of thesecriteria may not be necessary. Preferably, the solvent can solubilizethe starting material at least at a concentration of about 10% by weightor more, or about 15% by weight or more. More preferably, the solventcan solubilize the starting material at least at a concentration ofabout 20% by weight or more.

In addition, a suitable solvent should not react with the acylationreagents, i.e., the acid halide and the hindered base, or create anyunwanted side products with the starting material. Accordingly, choiceof a solvent depents on the choice of acid halide and hindered base, aswell as the functionality present on the starting material. Ideally, theonly reactive sites present on the starting material are the secondaryhydroxyl groups, thereby minimizing the possibility of unwantedreactions elsewhere. Examples of solvents include, among others,acetone, acetonitrile (CH₃CN), 1,2-dimethoxyethane (mono-glyme),dimethylformamide (DMF), tetrahydrofuran (TBF), and toluene.

Other reaction conditions that may be varied include the time andtemperature of the selective acylation reaction. In commercialembodiments, an increase of each of these parameters-increases the costof the reaction, i.e., the cost of making the end product. Thus,minimizing the time and temperature of the reaction is desirable and maybe achieved through the judicious choice of the above-discussedparameters.

For example, in a commercial process, a reaction time of less than about8 hours is preferred (e.g. representing an employee shift), withreaction times of about 6 hours or about 4 hours more preferred.Regarding the temperature of reaction, the closer it is to about ambienttemperature, the lower the amount of energy that needs to be used toeither heat or cool the reaction thereby making the process more costeffective. Moreover, temperature sensitivity of the starting materialand the end product should be taken into consideration when selectingthe selective acylating reagents and other reaction parameters andconditions.

In addition, a selective acylation reaction may be carried out atambient pressure in an atmospheric environment. Preferably the reactionis conducted in an inert atmosphere, e.g. nitrogen or argon. Inaddition, the reaction may be carried out at a pressure greater than orless than atmospheric pressure.

As with most chemical reactions, its progress may be monitored tocompleteness, e.g., using thin layer chromatography (TLC) or highperformance liquid chromatography (HPLC). In some embodiments, areaction may be deemed complete with about 90% of the end products isacylated at the desired hydroxyl group, e.g., at the C-2′ position of ataxane molecule, and/or about 5% or less of the starting material isunreacted. In other embodiments, a reaction may be deemed complete whenabout 95% of the end products is acylated at the desired hydroxyl groupand/or about 0.1% or less of the starting material is unreacted.Subsequent to completion of the selective acylation reaction or after aspecific reaction time, work up of the acylation reaction, and isolationand purification of the selectively acylated end product may beaccomplished using techniques readily known in the art.

For example, work up of selective acylation reaction conducted in CH₃CNtypically involves treating the reaction solution with a dilutehydrochloric acid (HCl) solution, partitioning with ethyl acetate(EtOAc), washing the organic layer with a dilute sodium bicarbonate(NaHCO₃) solution, washing the organic layer with a salt solution(brine), drying the organic layer with sodium sulfate (Na₂SO₄), andevaporating the organic layer to an amorphous solid. Subsequently, theamorphous solid may be crystallized using an appropriate solvent orsolvent system.

Because use of excess acid halide and hindered base preferred, polymericresins such as amino-functionalized resin beads or cation exchangeresins may be used in the work up of reaction to facilitate removal ofthe excess acylation reagents. The polymeric resins typically are addedto the reaction solution after completion of the reaction, then filteredout in the subsequent work up. Preferably an excess of the polymericresin is used compared to the acylation reagent to be removed. Forexample, a quantity of polymeric resin that is about 2 equivalents, orabout 4 equivalents is used. More preferably, about 6 equivalents ormore of the polymeric resin is used. Removal of an acid halide, e.g.,benzoyl chloride, may be realized by suing an amino-functionalized resinbead, e.g., Mitsubishi WA21J beads. Removal of a hindered base such as apyridine derivative, e.g., 2,6-lutidine, may be achieved using acationic exchange resin.

Alternatively, it was discovered that if the selective acylationreaction is conducted in THF with an acid chloride, much of thehydrochloric acid salt of the pyridine derivative formed during thecourse of the reaction may be removed by filtration. Although this maynot be possible when CH₃CN is the solvent as the hydrochloric acid saltof the pyridine derivative is soluble in CH₃CN. Subsequent precipitationof the crude end product in an excess of an appropriate solvent, e.g.,hexane or heptane, was found to remove much of the remaining hinderedbase, the acid chloride, and any anhydride that formed during reaction.

The end product of the selective acylation reaction may be analyzedusing analytical techniques known in the art such as infrared (IR)spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, e.g.,¹H-NMR and ¹³C-NMR, high performance liquid chromatography (HPLC), e.g.,reversed phase HPLC, and/or mass spectrometry (MS), e.g., electrosprayionization MS (ES-MS) and matrix-assisted laser desorption ionization MS(MALDI-MS). A combination of these techniques also may be used, e.g.,HPLC-MS.

In a preferred embodiment of the above-described methodology, theinvention is directed to methods of selectively acylating a taxanemolecule or mixtures of taxane molecules, e.g., from biomass or biomassextracts. Particularly, the method is directed to selectively acylatingthe hydroxyl group located at a C-2′ position of a taxane molecule whilethe hydroxyl group located at the C-7 position of the taxane moleculeremains unchanged. The method generally includes the steps of providinga solution comprising a taxane molecule in an organic solvent andcontacting the solution with a hindered base and an acid halide wherebythe hydroxyl group located at the C-2′ position is selectively acylated.It should understood that the above description of the general selectiveacylation reaction, including reagents, reaction parameters andconditions, and other information, applies equally to the selectiveacylation of a taxane molecule.

In the synthesis of taxane molecules, or in the conversion of taxanemolecules into other taxane molecules, it may be desirable toselectively acylate the C-2′ hydroxyl group of the taxane molecule whilemaintaining the C-7 hydroxyl group unprotected. For example, to meetGood Manufacturing Practice (GMP) standards, paclitaxel from varioussources can be selectively acylated with a benzoyl halide using methodsof the invention to produce a semi-synthetic intermediate which can betransformed back into paclitaxel. Provided that substantially all theoriginal paclitaxel was converted to the semi-synthetic intermediate,the resulting paclitaxel can be used in commercial pharmaceuticalpreparations. Thus, performing a selective acylation reaction where theC-7 hydroxyl group of a taxane molecule remains unprotected eliminatesadditional protection and deprotection steps, thereby reducing theoverall steps necessary in the synthetic scheme and providing a morecost effective methodology.

A selective acylation reaction also may be conducted using a mixture oftaxane molecules, each of which is selectively acylated at the desiredhydroxyl group, e.g., the C-2′ hydroxyl group. Mixtures of paclitaxel,cephalomannine, taxol C, and other taxane molecules occur in biomass orpartial separations or extracts from biomass. According to methods ofthe invention, two or more of paclitaxel, cephalomannine, taxol C, andother taxane molecules may be selectively acylated together in onereaction. Thus, the methods of the invention permit selective acylationof multiple taxane molecules in one step. Subsequently, the selectivelyacylated mixture of taxane molecules may be converted to a particulartaxane molecule determined by the choice of acid halide.

A preferred starting material is a taxane molecule having the formula:

where each of R₁, R₂, R₄, R₇, and R₁₀ independently is hydrogen, analkyl group, an acyl group, an aryl group, an arylalkyl group, a vinylgroup, an ether group, an ester group, a glycoside group, an oxo-group,or a hydroxyl protecting group, provided that one of R₂, R₇ and R₁₀ ishydrogen. Preferably, R₁ is hydrogen; R₂ is an acyl group or a hydroxylprotecting group; R₄ is an acetate group; R₇ is hydrogen, an alkylgroup, an aryl group, an ester group, an ether group, a glycoside group,an oxo-group, or a hydroxyl protecting group, and R₁₀ is hydrogen, analkyl group, an aryl group, an ester group, ether group, or a hydroxylprotecting group; R₁₀ is hydrogen, an alkyl group, an aryl group, anester group, an ether group, or a hydroxyl protecting group, providedthat one of R₂, R₇ and R₁₀ is hydrogen. More preferably, R₁ is ahydrogen, R₂ is a benzoyl group (PHC(O)—), R₄ is an acetate group(CH₃C(O)—), R₇ is hydrogen, and R₁₀ is an acetate group; or R₁ is ahydrogen, R₂ is a benzoyl group (PhC(O)—), R₄ is an acetate group(CH₃C(O)—), R₇ is hydrogen, and R₁₀ is hydrogen.

As stated above, Rx can be an amino group, a salt of an amino group, anamino group that is protected with an amino protecting group, or asubstituent which may be converted into an amino group. For example, Rxmay be an iminio group having the formula:

where RC is an alkyl group, an aryl group, an arylalkyl group, an vinylgroup, an ether group, or a heterocyclic group; and Z is a counterionsuch as chlorine (Cl⁻). While performing selective acylation, Rx ispreferably an amino group that is protected as an amide (—NHC(O)—), animine (—N═CH—), or a carbamate, (—NHC(O)O—).

More preferably, Rx is an imine group having the formula —N═CHRc, whereRc is an alkyl group, an aryl group, an arylalkyl group, an vinyl group,an ether group, or a heterocylic group; or Rx is an amide group havingthe formula —NHC(O)R_(N), where R_(N) is an alkyl group, an aryl group,an arylalkyl group, a vinyl group, an ether group, or a heterocylicgroup. Preferred Rc groups include, among others, aryl groups such asphenyl, alylalkyl groups such as benzyl, vinyl groups such as1-methyl-1-propenyl (CH₃CH═C(CH₃)—), alkyl groups such as n-pentyl,propyl, 1-methyl-propyl (CH₃CH₂CH(CH₃)—), and heterocyclic groups suchas 2-furanyl. Examples of R_(N) groups include, among others, acetyl(CH₃C(O)—), HOC(O)—, (CH₃OC(O)—, (CH₃CH(OH)C(OH)(CH₃)—) and PhNHC(O)—.Preferred R_(N) groups include, but are not limited to, phenyl,1-methyl-1-propenyl, n-pentyl, propyl, 1-methyl-propyl, benzyl, and2-furanyl as shown in Table 1.

A preferred end product of a selective acylation reaction with a taxanemolecule is a taxane molecule having the formula:

Another preferred end product of a selective acylation reaction with ataxane molecule is a taxane molecule having the formula:

Other preferred end products are the known taxane molecules depicted inTable 1 with a benzoyl group attached to the 2′-oxygen.

Preferably at least about 85% of the end product taxane molecule isselectively acylated at the C-2′ hydroxyl group without acylation at theC-7 hydroxyl group. Further, preferably less than about 1% of a bis- orhigher order acylated compound is formed and/or less than about 0.1% ofthe starting taxane molecule(s) remain unreacted.

In the selective acylation of a taxane molecule, preferred acid halidesare benzoyl chloride, tigloyl halide, hexanoyl chloride (caproylchloride), butyryl chloride, 2-methylbutyryl chloride, phenylacetylchloride, furoyl chloride, and tert-butyl chloroformate. Preferredhindered bases are trialkylamines having two of their alkyl groupslarger than ethyl, and pyridine derivatives that are substituted atleast at its 2- and 6-positions. Examples of preferred hindered basesare 2,4,6-collidine, 2,6-lutidine, NN-diisopropylethylamine,N-ethyldicyclohexylamine, and tripropylamine.

The quantity of each of the acid halide and hindered base is preferablyabout 4 or more equivalents based on the moles of starting taxanemolecule(s), and more preferably about 5 or more equivalents. Onepreferred embodiment employs about 5 equivalents of benzoyl chloride andabout 5.5 equivalents of 2,6-lutidine at about ambient temperature.

The preferred time of reaction is less than about 6 hours, and morepreferably less than about 4 hours. The preferred temperature of thereaction is less than about 40° C. and more preferably about ambienttemperature.

The preferred organic solvent for conducting a selective acylationreaction with a taxane molecule is THF. One reason for this choice isthat THF will typically solubilize certain taxane molecules at aconcentration of about 20% or greater. If THF is the organic solvent,the work up of the reaction is as described above. The preferred work upgenerally includes filtering the salt of the pyridine derivative andslowly adding the filtrate to about four or five times excess of hexaneor heptane which causes the end product taxane molecule(s) toprecipitate while the acylation reagents remain in solution. Heptane isthe preferred anti-solvent. It should be understood that a larger excessof an anti-solvent may be used, e.g., a ten-fold excess.

Optionally, the resulting taxane mixture of the present invention may bepurified using crystallization and/or one or more recrystallizations,depending on the desired purity of the end product taxane molecule.Crystallization and recrystallization is usually conducted using abinary or ternary solvent system, i.e., at least one solubilizingsolvent and at least one anti-solvent. Examples of solubilizing solventsmay include, among others, any halogenated hydrocarbon. Specificsolvents may also include acetone, methyl tert-butyl ether, methylenechloride, trifluorotoluene, and THF, for example. Examples ofanti-solvents, may include any hydrocarbon solvents such as, forexample, hexane and heptane. Examples of other solvent systems usefulwith taxane molecules may include, among others, any halogenatedhydrocarbon alone or combined with any hydrocarbon alkane,acetone/hexane, and methylene chloride/hexane. Methylene chloride/hexaneis preferred.

The resulting aceylated taxane molecule may be converted into a taxanemolecule in accordance with the processes described in U.S. Appl. No.60/370,583, filed Apr. 5, 2002. This application is incorporated hereinby reference in its entirety.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES

Solvents and reagents were purchased from Sigma-Aldrich Co. (Milwaukee,Wis.). ¹H NMR and ¹³C NMR chemical shifts are reported in parts permillion (ppm) relative to tetramethylsilane.

Example 1 Selective Acylation of Paclitaxel with Benzoyl Chloride and2.6-Lutidine or 2,4,6-Collidine

A 25% (w/v) solution of paclitaxel in anhydrous tetrahydrofuran (THF)was prepared. 0.5 mL aliquots of this solution (0.148 mmol) were addedto small vials, and treated with 5.5 equivalents of hindered basefollowed by 5.0 equivalents of benzoyl chloride. The reactions werestirred at room temperature for 4 hours and sampled for HPLC analysisusing the HPLC protocol described below. The 2′-O-benzoyl paclitaxel(2′-OBz paclitaxel) was substantially identical to that reported byKingston et al, in U.S. Pat. Nos. 5,319,112 and 5,470,866. The2′,7-bis-O-benzoyl paclitaxel (2′,7-bis-OBz paclitaxel) was isolated andidentified by ¹H NMR and ¹³C NMR. Table 2 shows the percentages ofstarting material and major products as determined by HPLC analysis.TABLE 2 % 2′,7-bis-OBZ Base % Paclitaxel % 2′OBZ Paclitaxel Paclitaxel2,6-Lutidine 0.43 99.44 0.12 2,4,6-Collidine 0.52 99.31 0.17

HPLC Method: Column: Supelco Diphenyl, 150×4.6 mm, 5 micron. Flow Rate:1.5 mL/min. Gradient: Start at 70%/30% water/acetonitrile (CH₃CN) with alinear gradient reaching 50%/50% water/CH₃CN at 10 minutes, then alinear gradient reaching 20%/80% water/CH₃CN at 13 minutes, then holduntil 16 minutes. Re-equilibration is with 70%/30% water/CH₃CN for 4minutes. Detection: 227 and 254 nm.

2′,7-Bis-O-Benzoyl Paclitaxel NMR Results

¹H NMR, 400 MHz (CDCl₃): 1.18 s 3H, 1.20 s 3H, 1.80 m 1H, 1.95 s 6H,2.06 br s 3H, 2.15 m 1H, 2.37 m 1H, 2.45 s 3H, 2.79 m 1H, 4.05 d (6.8Hz) 1H, 4.23 d (8.0 Hz) 1H, 4.34 d (8.0 Hz) 1H, 5.00 d (8.4 Hz) 1H,5.73-5.80 m 3H, 6.05 dd (3.6, 8.8 Hz) 1H, 6.24 t (7.6 Hz) 1H, 6.43 s 1H,7.16 d (9.2 Hz) 1H, 7.31-8.13 m 25H.

¹³C NMR 100 MHz (CDCl₃): 11.04, 14.50, 20.36,21.33, 22.51, 26.35, 33.81,35.42, 43.18, 46.58, 53.32, 56.15, 71.81, 72.02, 74.64, 74.73, 76.29,78.79, 80.71, 83.96, 126.68, 127.02, 128.04, 128.52, 128.67,128.68,129.05, 129.10, 129.73, 129.82, 130.13, 131.91, 132.66, 133.67,133.72, 133.83, 136.86, 141.26, 156.75, 165.13, 165.48, 166.89, 167.14,168.22, 168.24, 169.53, 202.52.

Example 2 Selective Acylation of Cephalomannine with Benzoyl Chlorideand 2.6-Lutidine

A 25-mL flask was thoroughly dried with a heat gun and cooled by passingnitrogen through it. Cephalomannine (2.0 g, 2.41 mmol) was added,followed by anhydrous THF (10 mL). Within several minutes after stirringat room temperature, the solution was homogeneous. Next, 2,6-lutidine(1.6 ml, 5.5 eq.) was added at once and stirred for about 2 minutes.Benzoyl chloride (1.4 mL, 5.0 eq.) was added over 2-3 minutes. About 5minutes after the addition of the acylation reagents was complete, thestirred solution became heterogeneous. HPLC assays were performed atvarious intervals and at about 4 hours, the reaction was deemedcomplete.

The 2,6-lutidine-HCl was filtered off through a sintered glass funneland washed with about 5 mL of anhydrous THF. The combined filtrate wasadded to about 60 mL of heptane over about 5 minutes, causingprecipitation. The suspension was transferred to a Buchner funnel andthe collected yellow solid was washed three times with about 5 mL of a4:1 heptane:THF solution. The yellow solid was then added to about 10 mLof methylene chloride (CH₂Cl₂) and stirred for about 10 minutes. Then,about 25 mL of heptane was added over about 10 minutes. After siring forabout 10 minutes, the crystalline solid was filtered through a Buchnerfunnel and washed two times with about 5 mL of a 2.5:1 heptane: CH₂Cl₂solution. After drying for about 2 days under vacuum at about 40-50° C.,the crystalline solid weighed 2.06 grams and contained 2.00 g2′-O-benzoyl cephalomannine (2′-OBz cephalomannine) (88.9% yield), 0.3%unreacted cephalomannine, and no detectable 2′,7-bis-O-benzoylcephalomannine as determined by HPLC.

2′-OBz Cephalomannine NMR Results

¹H NMR, 400 MHz (CDCl₃): 1.14 s 3H, 1.26 s 3H, 1.68 s 3H, 1.72 dd (1.2,7.2 Hz) 3H, 1.82 br s3H, 1.87 m 1H, 1.95 d (1.2 Hz) 3H, 2.15 m 1H, 2.23s 3H, 2.31 m 1H, 2.41 s 3H, 2.50 m 1H, 3.81 d (6.8 Hz) 1H, 4.18 d (8.4Hz) 1H, 4.30 d (8.4 Hz) 1H, 4.45 dd (6.8, 9.6 Hz) 1H, 4.96 dd (2.0, 9.2Hz) 1H, 5.62 d (4.0 Hz) 1H, 5.68 d (6.8 Hz) 1H, 5.90 dd (4.0, 8.8 Hz)1H, 6.26 t (7.6) 1H, 6.30 s 1H, 6.44 m 1H, 6.59 d (8.8 Hz) 1H, 7.30-8.12m 15H.

¹³C NMR 100 MHz (CDCl₃): 9.58, 12.44, 13.98, 14.86, 20.81, 22.16, 22.61,26.81, 35.48, 35.49, 43.15, 45.54, 52.75, 58.48, 71.80, 72.11, 74.73,75.10, 75.59, 76.40, 79.27, 80.96, 84.43, 126.58, 128.39, 128.66,128.70, 129.00, 129.13, 129.79, 130.18, 131.51, 131.70, 132.63, 133.66,133.86, 137.19, 142.98, 165.58, 167.06, 168.14, 168.76, 169.77, 171.25,203.83.

Example 3 Survey of Hindered Bases in the Selective Acylation ofPaciltaxel with 2-Furoyl Chloride

A 25% (w/v) solution of paclitaxel in anhydrous THF was prepared. 0.5 mLaliquots of this solution (0.148 millimoles) were added to small vials,and treated with 5.5 equivalents of a hindered base followed by 5.0equivalents of 2-furoyl chloride. The reactions were stirred at roomtemperature for 4 hours, and sampled for HPLC analysis using the HPLCprotocol described in Example 1. Table 3 shows the percentages ofstarting material and major products as determined by HPLC analysis.TABLE 3 % 2′,7-bis-O- % 2′-0-Furoyl Furoyl Base % Paclitaxel PaclitaxelPaclitaxel 2,3-Lutidine 0 86.43 13.57 2,4-Lutidine 0.64 88.10 11.262,5-Lutidine 0.15 84.55 15.30 2,6-Lutidine 0.18 99.57 0.26 3,4-Lutidine0.23 1.95 97.82 3,5-Lutidine 0 0.04 99.96 2,3,5-Collidine 0.16 79.7620.08 2,4,6-Collidine 0.21 98.91 0.89 Pyridine 0 0.13 99.87 2-PhenylPyridine 67.05 32.77 0.18 N-Ethyldicyclohexyl 0 99.95 0.05 amineN,N-Diisopropyl- 39.00 60.78 0.23 isobutylamine N,N-diisopropylethyl- 099.88 0.16 amine Triethylamine 0 88.87 11.13 Tripropylamine 0 99.22 0.78Imidazole 0.17 93.29 6.54 Diazabicyclooctane 0 91.00 9.00 (DABCO)4-(N,N-Dimethylamino)- 0 0.18 99.92 pyridine (DMAP) Diazabicycloundecane20.37 69.84 9.79 (DBU)

Example 4 Selective Acylation of Mixed Taxanes at Large Scale

A 5-L flask was thoroughly dried with a heat gun and cooled by passingnitrogen through it. A taxane mixture containing 89.5% taxane molecules(consisting of 11.6% paclitaxel (taxol A), 21.4% cephalomannine (taxolB), 43.7% taxol C, 0.2% taxol D, 3.5% taxol E, 8.7% taxol F, and 0.4%taxol G) (753.2 g, ˜0.88 mol) was added, followed by anhydrous THF (3.0L). Within several minutes after stirring at room temperature, thesolution was homogeneous. Next, 2,6-lutidine (565 mL, 5.5 eq.) was addedat once and stirred for about 2 minutes. Benzoyl chloride (510 mL, 5.0eq.) was added over about 10 minutes (no significant exotherm wasexperienced). About 1 minute after the addition of the acylationreagents was complete, the stirred solution became heterogeneous. HPLCassays were performed at various intervals and at about 6 hours, thereaction was deemed complete.

The 2,6-lutidine-HCl was filtered off through a sintered glass funneland washed with about 500 mL of anhydrous ThF. One-half of the combinedfiltrate was added to about 12 L of heptane over about 15 minutes,causing precipitation. This precipitate was filtered using a Buchnerfunnel. The other half of the filtrate was processed likewise. Thecollected yellow solid was washed twice with a total of about 3.75 L ofa 4:1 heptane: THF solution. The yellow solid was then added to about 3L of CH₂Cl₂ and stirred for about 10 minutes to yield a crystallinesolid. Then, 6 L of heptane was added over about 10 minutes withstirring. After stirring for about 30 minutes, the crystalline solid wasfiltered using a Buchner funnel and washed three times with a total ofabout 6 L of a 3:1 heptane:CH₂Cl₂ solution. After drying for about 60hours under vacuum at about 40-50° C.; the crystalline solid weighed 728grams and contained 645.9 g 2-O′-benzoyl taxane molecules (85.4% yield),and less than 0.5% unreacted taxane molecules and 2′,7-bis-O-benzoyltaxane molecules as determined by HPLC.

Incorporation by Reference

The content of each of the patent and non-patent documents referred toherein is expressly incorporated herein by reference.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A method of selectively acylating a compound comprising at least afirst and second secondary hydroxyl groups, the method comprising thesteps of (a) providing a solution of the compound in a solvent; and (b)contacting the solution with a hindered base and an acylating agentthereby to selectively acylate the first or secondary hydroxyl group. 2.The method of claim 1 wherein the compound is a taxane molecule.
 3. Themethod of claim 1, wherein the acylating agent is an acid halide.
 4. Themethod of claim 1, wherein the acid halide is an acid chloride.
 5. Themethod of claim 1 wherein the acid halide is selected from the groupconsisting of benzoyl halide, tigloyl halide, hexanoyl halide, butyrylhalide, 2-methylbutyryl halide, phenylacetyl halide, furoyl halide, andtert-butyl haloformate.
 6. The method of claim 1 wherein the hinderedbase is a pyridine derivative or a trialkylamine.
 7. The method of claim5, wherein the trialkylamine is N-ethyldicyclohexylamine orN,N-diisopropylethylamine.
 8. The method of claim 4 wherein the pyridinederivative is selected from the group consisting of 2,6-lutidine, and2,4,6-collidine.
 9. A method of selectively acylating a hydroxyl grouplocated at a C-2′ position of a taxane molecule having an unprotectedhydroxyl group located at a C-7 position, the method comprising thesteps of: (a) providing a solution comprising a taxane molecule in anorganic solvent; and (b) contacting the solution with a hindered baseand an acylating agent hereby to selectively acylate the hydroxyl grouplocated at the C-2′ position.
 10. The method of claim 9, wherein theacylating agent is an acid halide.
 11. The method of claim 9 wherein theacid halide is an acid chloride.
 12. The method of claim 11 wherein theacid chloride is selected from the group consisting of benzoyl chloride,tigloyl chloride, hexanoyl chloride, butyryl chloride, 2-methylbutyrylchloride, phenylacetyl chloride, furoyl chloride, and tert-butylchloroformate.
 13. The method of claim 12 wherein the acid chloride isbenzoyl chloride.
 14. The method of claim 12 wherein the acid chlorideis tigloyl chloride.
 15. The method of claim 11 wherein the hinderedbase is a pyridine derivative or a trialkylamine.
 16. The method ofclaim 15 wherein the pyridine derivative is selected from the groupconsisting of 2,6-lutidine, and 2, 4, 6-collidine.
 17. The method ofclaim 15 wherein the trialkylamine is N-ethyldicyclohexylamine orN,N-diisopropylethylamine.
 18. The method of claim 9 wherein the organicsolvent is tetrahydrofuran.
 19. The method of claim 9 wherein theorganic solvent solubilizes the taxane molecule at a concentration of atleast about 15% by weight.
 20. The method of claim 9 wherein selectiveacylation occurs in about 6 hours or less.
 21. The method of claim 9wherein selective acylation occurs at a temperature of about 40° C. orless.
 22. The method of claim 9 wherein selective acylation occurs atabout ambient temperature.
 23. The method of claim 9 wherein each of thehindered base and the acid halide are present in an amount greater thanor equal to about 4 equivalents of the taxane molecule.
 24. The methodof claim 9, wherein the taxane molecule has the formula:

wherein R₁ is hydrogen; R₂ is hydrogen, an acyl group or a hydroxylprotecting group; R₄ is an acetate group; R₇ is hydrogen, an alkylgroup, an aryl group, an ester group, an ether group, a glycoside group,an oxo group, or a hydroxyl protecting group; R₁₀ is hydrogen; and Rx isan amino group, a salt of an amino group, or an amino group that isprotected with an amino protecting group.
 25. The method of claim 24wherein Rx is N═CHRc or —NHC(O)R_(n), wherein Rc is an alkyl group, anaryl group, an arylalkyl group, an vinyl group, or an ether group; andR_(n) is an alkyl group, an aryl group, an arytalkyl group, a vinylgroup, or an ether group.
 26. The method of claim 25 wherein Rc isselected from the group consisting of phenyl, 1-methyl-1-propenyl,n-pentyl, propyl, 1-methyl-propyl, benzyl, and 2-furanyl.
 27. The methodof claim 25 wherein RN is selected from the group consisting of phenyl;1-methyl-1-propenyl, n-pentyl, propyl, 1-methyl-propyl, benzyl,2-furanyl, and tert-butoxy.
 28. A method of selectively acylating ataxane molecule, the method comprising the steps of (a) providing asolution of tetrahydrofuran and a taxane molecule having the formula:

wherein R₁ is hydrogen; R₂ is a benzoyl group; R4 is an acetate group;R₇ is hydrogen; R₁₀ is hydrogen or an acetate group; and Rx is N═CHRc or—NHC(O)R_(N), wherein Rc is an alkyl group, an aryl group, an arylalkylgroup, an vinyl group, or an ether group; and Rn is an alkyl group, anaryl group, an arylalkyl group, a vinyl group, or an ether group; and(b) adding 2,6-lutidine or N ethyldicyclohexylamine and an acid chlorideto the solution thereby to selectively acylate the hydroxyl grouplocated at the C-2′ position.
 29. The method of claim 28 wherein R₁₀ ishydrogen.
 30. The method of claim 28 wherein R₁₀ is an acetate group.31. The method of claim 29 wherein Rx is N═CHRc, and Rc is selected fromthe group consisting of phenyl, 1-methyl-1-propenyl, n-pentyl, propyl,1-methyl-propyl, benzyl; 2-furanyl, and tert-butoxy.
 32. The method ofclaim 29 wherein Rx is —NHC(O)R_(n), and R_(n) is selected from thegroup consisting of phenyl, 1-methyl-1-propenyl, n-pentyl, propyl,1-methyl-propyl, benzyl, 2-furanyl, and tert-butoxy.
 33. The method ofclaim 30 wherein Rx is —N═CHR_(c) and R_(n) is selected from the groupconsisting of phenyl, 1-methyl-1-propenyl, n-pentyl, propyl,1-methyl-propyl, benzyl, 2-furanyl, and tert-butoxy.
 34. The method ofclaim 30 wherein Rx is —NHC(O)RN, and RN is selected from the groupconsisting of phenyl, 1-methyl-1-propenyl, n-pentyl, propyl,1-methyl-propyl, benzyl, 2-furanyl, and tert-butoxy.
 35. The method ofclaim 31 wherein the acid chloride is selected from the group consistingof benzoyl chloride, tigloyl chloride, hexanoyl chloride, butyrylchloride, 2-methylbutyryl chloride, phenylacetyl chloride, furoylchloride, and tert-butyl chloroformate.
 36. The method of claim 32wherein the acid chloride is selected from the group consisting ofbenzoyl chloride, tigloyl chloride, hexanoyl chloride, butyryl chloride,2-methylbutyryl chloride, phenylacetyl chloride, furoyl chloride, andtert-butyl chloroformate.
 37. The method of claim 33 wherein the acidchloride is selected from the group consisting of benzoyl chloride,tigloyl chloride, hexanoyl chloride, butyryl chloride, 2-methylbutyrylchloride, phenylacetyl chloride, furoyl chloride, and tert-butylchloroformate.
 38. The method of claim 34 wherein the acid chloride isselected from the group consisting of benzoyl chloride, tigloylchloride, hexanoyl chloride, butyryl chloride, 2-methylbutyryl chloride,phenylacetyl chloride, furoyl chloride, and tert-butyl chloroformate.39. The method of claim 28 wherein R₁₀ is an acetate group, Rx is—NHC(O)R_(n), wherein R_(n) is phenyl, and the acid chloride is benzoylchloride.
 40. The method of claim 28 wherein R₁₀ is an acetate group, Rxis —NHC(O)R_(n), wherein R_(n) is 1-methyl-1-propenyl, and the acidchloride is benzoyl chloride.
 41. The method of claim 28 wherein R_(n10)is an acetate group, Rx is —NHC(O)R_(n), wherein R_(n), wherein R_(n) isn-pentyl, and the acid chloride is benzoyl chloride.
 42. The method ofclaims 1,9, or 28 further comprising the step of crystallizing theacylated compound with at least one solubilizing solvent and optionallyat least one anti-solvent.
 43. The method of claim 42, wherein thesolvent is a halogenated hydrocarbon.
 44. The method of claim 42,wherein the solubilizing solvent is selected form the group consistingof acetone, methyl tert-butyl ether, triflourotoluene or THF.
 45. Themethod of claim 42, wherein the solubilizing solvent is methylenechloride.
 46. The method of claim 42, wherein the solvent is methylenechloride and the antisolvent is hexane.
 47. The method of claim 42,wherein the antisolvent is a hydrocarbon alkane.